A model for cofactor use during HIV-1 reverse transcription and nuclear entry

ABSTRACTThe HIV-1 capsid protein (CA) facilitates reverse transcription and nuclear entry of the virus. However, CA’s role in post-nuclear entry steps remains speculative. We describe a direct link between CA and integration by employing the capsid inhibitor PF74 as a probe coupled with the biochemical analysis of HIV-1 preintegration complexes (PICs) isolated from acutely infected cells. At a low micromolar concentration, PF74 potently inhibited HIV-1 infection without affecting reverse transcription. Surprisingly, PF74 markedly reduced proviral integration owing to inhibition of nuclear entry and/or integration. However, a 2-fold reduction in nuclear entry by PF74 did not quantitatively correlate with the level of antiviral activity. Titration of PF74 against the integrase inhibitor raltegravir showed an additive antiviral effect that is dependent on a block at the post-nuclear entry step. PF74’s inhibitory effect was not due to the formation of defective viral DNA ends or a delay in integration, suggesting that the compound inhibits PIC-associated integration activity. Unexpectedly, PICs recovered from cells infected in the presence of PF74 exhibited elevated integration activity. PF74’s effect on PIC activity is CA specific since the compound did not increase the integration activity of PICs of a PF74-resistant HIV-1 CA mutant. Sucrose gradient-based fractionation studies revealed that PICs assembled in the presence of PF74 contained lower levels of CA, suggesting a negative association between CA and PIC-associated integration activity. Finally, the addition of a CA-specific antibody or PF74 inhibited PIC-associated integration activity. Collectively, our results demonstrate that PF74’s targeting of PIC-associated CA results in impaired HIV-1 integration.IMPORTANCEAntiretroviral therapy (ART) that uses various combinations of small molecule inhibitors has been highly effective in controlling HIV. However, the drugs used in the ART regimen are expensive, cause side effects, and face viral resistance. The HIV-1 CA plays critical roles in the virus life cycle and is an attractive therapeutic target. While currently there is no CA-based therapy, highly potent CA-specific inhibitors are being developed as a new class of antivirals. Efforts to develop a CA-targeted therapy can be aided through a clear understanding of the role of CA in HIV-1 infection. CA is well established to coordinate reverse transcription and nuclear entry of the virus. However, the role of CA in post-nuclear entry steps of HIV-1 infection is poorly understood. We show that a CA-specific drug PF74 inhibits HIV-1 integration revealing a novel role of this multifunctional viral protein in a post-nuclear entry step of HIV-1 infection.

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HIV-1 requires capsid remodelling at the nuclear pore for nuclear entry and integration

10.1101/2021.03.18.436028 ◽

2021 ◽

Author(s):

Anabel Guedán ◽

Callum D Donaldson ◽

Ophélie Cosnefroy ◽

Ian A Taylor ◽

Kate N. Bishop

Keyword(s):

Reverse Transcription ◽

Integration Site ◽

Productive Infection ◽

Nuclear Pore ◽

Virus Infectivity ◽

Nuclear Speckles ◽

Nuclear Entry ◽

The Core ◽

Fold Reduction ◽

Hiv 1

The capsid (CA) lattice of the HIV-1 core plays a key role during infection. From the moment the core is released into the cytoplasm, it interacts with a range of cellular factors that, ultimately, direct the pre-integration complex to the integration site. For integration to occur, the CA lattice must disassemble. Early uncoating or a failure to do so has detrimental effects on virus infectivity, indicating that an optimal stability of the viral core is crucial for infection. Here, we introduced cysteine residues into HIV-1 CA in order to induce disulphide bond formation and engineer hyper-stable mutants that are slower or unable to uncoat, and then followed their replication. From a panel of mutants, we identified three with increased capsid stability in cells and found that, whilst the M68C/E212C mutant had a 5-fold reduction in reverse transcription, two mutants, A14C/E45C and E180C, were able to reverse transcribe to approximately WT levels. Moreover, these mutants only had a 5-fold reduction in 2-LTR circle production, suggesting that not only could reverse transcription complete in hyper-stable cores, but that the nascent viral cDNA could enter the nuclear compartment. Furthermore, we observed significant levels of A14C/E45C mutant capsid in nuclear and chromatin-associated fractions implying that the hyper-stable cores themselves entered the nucleus. Immunofluorescence studies revealed that although the A14C/E45C mutant capsid reached the nuclear pore with the same kinetics as wild type capsid, it was then retained at the pore in association with Nup153. Crucially, infection with the hyper-stable mutants did not promote CPSF6 re-localisation to nuclear speckles, despite the mutant capsids being competent for CPSF6 binding. These observations suggest that hyper-stable cores are not able to uncoat, or remodel, enough to pass through or dissociate from the nuclear pore and integrate successfully. This, is turn, highlights the importance of capsid lattice flexibility for nuclear entry. In conclusion, we hypothesise that during a productive infection, a capsid remodelling step takes place at the nuclear pore that releases the core complex from Nup153, and relays it to CPSF6, which then localises it to chromatin ready for integration.

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Lv2, a Novel Postentry Restriction, Is Mediated by both Capsid and Envelope

Journal of Virology ◽

10.1128/jvi.78.4.2006-2016.2004 ◽

2004 ◽

Vol 78 (4) ◽

pp. 2006-2016 ◽

Cited By ~ 40

Author(s):

Christian Schmitz ◽

David Marchant ◽

Stuart J. D. Neil ◽

Keith Aubin ◽

Sandra Reuter ◽

...

Keyword(s):

Life Cycle ◽

Reverse Transcription ◽

Site Directed Mutagenesis ◽

Single Amino Acid ◽

Nuclear Entry ◽

Cellular Compartment ◽

Glycoprotein G ◽

Reverse Transcription Step ◽

Viral Determinants ◽

Hiv 1

ABSTRACT The characterization of restrictions to lentivirus replication in cells identifies critical steps in the viral life cycle and potential therapeutic targets. We previously reported that a human immunodeficiency virus type 2 (HIV-2) isolate was restricted to infection in some human cells, which led us to identify a step in the life cycle of HIV-2 detected after reverse transcription but prior to nuclear entry. The block is bypassed with a vesicular stomatitis virus glycoprotein G (VSV-G) envelope (A. McKnight et al., J. Virol. 75:6914-6922, 2001). We hypothesized that, although the restriction is apparent at a post-reverse transcription step, the lack of progress results from a failure of the virus to reach a cellular compartment with access to the nucleus. Here we analyzed molecular clones of the restricted virus, MCR, and an unrestricted virus, MCN. Using sequence analysis and gene swapping, we mapped the viral determinants to gag and env. Site-directed mutagenesis identified a single amino acid at position 207 in CA to be responsible for the gag restriction. Pseudotype experiments indicate that this step is also important for the infection of cells by HIV-1. The HIV-1 NL4.3 core is restricted if supplied with a restricted MCR envelope but not with VSV-G. Also the NL4.3 envelope rescues the restricted core of HIV-2 MCR. Abrogation experiments with MLV demonstrate that the restriction is distinct from Fv1/Ref1/Lv1. We propose that this represents a new lentiviral restriction, Lv2. Thus, the envelope and capsid of HIV act to ensure that the virus is delivered into an appropriate cellular compartment that allows postentry events in viral replication to proceed efficiently.

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HIV-1 requires capsid remodelling at the nuclear pore for nuclear entry and integration

PLoS Pathogens ◽

10.1371/journal.ppat.1009484 ◽

2021 ◽

Vol 17 (9) ◽

pp. e1009484

Author(s):

Anabel Guedán ◽

Callum D. Donaldson ◽

Eve R. Caroe ◽

Ophélie Cosnefroy ◽

Ian A. Taylor ◽

...

Keyword(s):

Reverse Transcription ◽

Integration Site ◽

Productive Infection ◽

Nuclear Pore ◽

Virus Infectivity ◽

Nuclear Speckles ◽

Nuclear Entry ◽

The Core ◽

Fold Reduction ◽

Hiv 1

The capsid (CA) lattice of the HIV-1 core plays a key role during infection. From the moment the core is released into the cytoplasm, it interacts with a range of cellular factors that, ultimately, direct the pre-integration complex to the integration site. For integration to occur, the CA lattice must disassemble. Early uncoating or a failure to do so has detrimental effects on virus infectivity, indicating that an optimal stability of the viral core is crucial for infection. Here, we introduced cysteine residues into HIV-1 CA in order to induce disulphide bond formation and engineer hyper-stable mutants that are slower or unable to uncoat, and then followed their replication. From a panel of mutants, we identified three with increased capsid stability in cells and found that, whilst the M68C/E212C mutant had a 5-fold reduction in reverse transcription, two mutants, A14C/E45C and E180C, were able to reverse transcribe to approximately WT levels in cycling cells. Moreover, these mutants only had a 5-fold reduction in 2-LTR circle production, suggesting that not only could reverse transcription complete in hyper-stable cores, but that the nascent viral cDNA could enter the nuclear compartment. Furthermore, we observed A14C/E45C mutant capsid in nuclear and chromatin-associated fractions implying that the hyper-stable cores themselves entered the nucleus. Immunofluorescence studies revealed that although the A14C/E45C mutant capsid reached the nuclear pore with the same kinetics as wild type capsid, it was then retained at the pore in association with Nup153. Crucially, infection with the hyper-stable mutants did not promote CPSF6 re-localisation to nuclear speckles, despite the mutant capsids being competent for CPSF6 binding. These observations suggest that hyper-stable cores are not able to uncoat, or remodel, enough to pass through or dissociate from the nuclear pore and integrate successfully. This, is turn, highlights the importance of capsid lattice flexibility for nuclear entry. In conclusion, we hypothesise that during a productive infection, a capsid remodelling step takes place at the nuclear pore that releases the core complex from Nup153, and relays it to CPSF6, which then localises it to chromatin ready for integration.

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Cyclophilin A promotes HIV-1 reverse transcription but its effect on transduction correlates best with its effect on nuclear entry of viral cDNA

Retrovirology ◽

10.1186/1742-4690-11-11 ◽

2014 ◽

Vol 11 (1) ◽

Cited By ~ 61

Author(s):

Alberto De Iaco ◽

Jeremy Luban

Keyword(s):

Reverse Transcription ◽

Cyclophilin A ◽

Nuclear Entry ◽

Viral Cdna ◽

Hiv 1

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Quantitative microscopy of functional HIV post-entry complexes reveals association of replication with the viral capsid

eLife ◽

10.7554/elife.04114 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 107

Author(s):

Ke Peng ◽

Walter Muranyi ◽

Bärbel Glass ◽

Vibor Laketa ◽

Stephen R Yant ◽

...

Keyword(s):

Reverse Transcription ◽

Nuclear Import ◽

Viral Proteins ◽

Host Factors ◽

Quantitative Detection ◽

Quantitative Microscopy ◽

Nuclear Entry ◽

High Concentrations ◽

Post Entry ◽

Hiv 1

The steps from HIV-1 cytoplasmic entry until integration of the reverse transcribed genome are currently enigmatic. They occur in ill-defined reverse-transcription- and pre-integration-complexes (RTC, PIC) with various host and viral proteins implicated. In this study, we report quantitative detection of functional RTC/PIC by labeling nascent DNA combined with detection of viral integrase. We show that the viral CA (capsid) protein remains associated with cytoplasmic RTC/PIC but is lost on nuclear PIC in a HeLa-derived cell line. In contrast, nuclear PIC were almost always CA-positive in primary human macrophages, indicating nuclear import of capsids or capsid-like structures. We further show that the CA-targeted inhibitor PF74 exhibits a bimodal mechanism, blocking RTC/PIC association with the host factor CPSF6 and nuclear entry at low, and abrogating reverse transcription at high concentrations. The newly developed system is ideally suited for studying retroviral post-entry events and the roles of host factors including DNA sensors and signaling molecules.

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The Role of Capsid in HIV-1 Nuclear Entry

Viruses ◽

10.3390/v13081425 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1425

Author(s):

Anabel Guedán ◽

Eve R. Caroe ◽

Genevieve C. R. Barr ◽

Kate N. Bishop

Keyword(s):

Nuclear Pore ◽

Outer Face ◽

Nuclear Entry ◽

Nuclear Compartment ◽

Critical Step ◽

Technological Advances ◽

Dividing Cells ◽

Hiv 1 ◽

Gain Access

HIV-1 can infect non-dividing cells. The nuclear envelope therefore represents a barrier that HIV-1 must traverse in order to gain access to the host cell chromatin for integration. Hence, nuclear entry is a critical step in the early stages of HIV-1 replication. Following membrane fusion, the viral capsid (CA) lattice, which forms the outer face of the retroviral core, makes numerous interactions with cellular proteins that orchestrate the progress of HIV-1 through the replication cycle. The ability of CA to interact with nuclear pore proteins and other host factors around the nuclear pore determines whether nuclear entry occurs. Uncoating, the process by which the CA lattice opens and/or disassembles, is another critical step that must occur prior to integration. Both early and delayed uncoating have detrimental effects on viral infectivity. How uncoating relates to nuclear entry is currently hotly debated. Recent technological advances have led to intense discussions about the timing, location, and requirements for uncoating and have prompted the field to consider alternative uncoating scenarios that presently focus on uncoating at the nuclear pore and within the nuclear compartment. This review describes recent advances in the study of HIV-1 nuclear entry, outlines the interactions of the retroviral CA protein, and discusses the challenges of investigating HIV-1 uncoating.

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SUMOylation of SAMHD1 at Lysine 595 is required for HIV-1 restriction in non-cycling cells

Nature Communications ◽

10.1038/s41467-021-24802-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Charlotte Martinat ◽

Arthur Cormier ◽

Joëlle Tobaly-Tapiero ◽

Noé Palmic ◽

Nicoletta Casartelli ◽

...

Keyword(s):

Antiviral Activity ◽

Reverse Transcription ◽

Immune Cells ◽

Viral Restriction ◽

Antiviral Function ◽

Hiv 1 ◽

Constitutively Active

AbstractSAMHD1 is a cellular triphosphohydrolase (dNTPase) proposed to inhibit HIV-1 reverse transcription in non-cycling immune cells by limiting the supply of the dNTP substrates. Yet, phosphorylation of T592 downregulates SAMHD1 antiviral activity, but not its dNTPase function, implying that additional mechanisms contribute to viral restriction. Here, we show that SAMHD1 is SUMOylated on residue K595, a modification that relies on the presence of a proximal SUMO-interacting motif (SIM). Loss of K595 SUMOylation suppresses the restriction activity of SAMHD1, even in the context of the constitutively active phospho-ablative T592A mutant but has no impact on dNTP depletion. Conversely, the artificial fusion of SUMO2 to a non-SUMOylatable inactive SAMHD1 variant restores its antiviral function, a phenotype that is reversed by the phosphomimetic T592E mutation. Collectively, our observations clearly establish that lack of T592 phosphorylation cannot fully account for the restriction activity of SAMHD1. We find that SUMOylation of K595 is required to stimulate a dNTPase-independent antiviral activity in non-cycling immune cells, an effect that is antagonized by cyclin/CDK-dependent phosphorylation of T592 in cycling cells.

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High-resolution view of HIV-1 reverse transcriptase initiation complexes and inhibition by NNRTI drugs

Nature Communications ◽

10.1038/s41467-021-22628-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Betty Ha ◽

Kevin P. Larsen ◽

Jingji Zhang ◽

Ziao Fu ◽

Elizabeth Montabana ◽

...

Keyword(s):

Reverse Transcriptase ◽

Viral Entry ◽

Reverse Transcription ◽

Molecular Mechanisms ◽

Dissociation Kinetics ◽

Structural Mechanism ◽

Medium Resolution ◽

Kinetics Of ◽

Hiv 1

AbstractReverse transcription of the HIV-1 viral RNA genome (vRNA) is an integral step in virus replication. Upon viral entry, HIV-1 reverse transcriptase (RT) initiates from a host tRNALys3 primer bound to the vRNA genome and is the target of key antivirals, such as non-nucleoside reverse transcriptase inhibitors (NNRTIs). Initiation proceeds slowly with discrete pausing events along the vRNA template. Despite prior medium-resolution structural characterization of reverse transcriptase initiation complexes (RTICs), higher-resolution structures of the RTIC are needed to understand the molecular mechanisms that underlie initiation. Here we report cryo-EM structures of the core RTIC, RTIC–nevirapine, and RTIC–efavirenz complexes at 2.8, 3.1, and 2.9 Å, respectively. In combination with biochemical studies, these data suggest a basis for rapid dissociation kinetics of RT from the vRNA–tRNALys3 initiation complex and reveal a specific structural mechanism of nucleic acid conformational stabilization during initiation. Finally, our results show that NNRTIs inhibit the RTIC and exacerbate discrete pausing during early reverse transcription.

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Extended Interactions between HIV-1 Viral RNA and tRNALys3 Are Important to Maintain Viral RNA Integrity

International Journal of Molecular Sciences ◽

10.3390/ijms22010058 ◽

2020 ◽

Vol 22 (1) ◽

pp. 58

Author(s):

Thomas Gremminger ◽

Zhenwei Song ◽

Juan Ji ◽

Avery Foster ◽

Kexin Weng ◽

...

Keyword(s):

Reverse Transcription ◽

Potential Interaction ◽

Viral Rna ◽

Primer Binding Site ◽

Rna Integrity ◽

Immunodeficiency Virus ◽

Infected Cells ◽

Extended Interaction ◽

Hiv 1

The reverse transcription of the human immunodeficiency virus 1 (HIV-1) initiates upon annealing of the 3′-18-nt of tRNALys3 onto the primer binding site (PBS) in viral RNA (vRNA). Additional intermolecular interactions between tRNALys3 and vRNA have been reported, but their functions remain unclear. Here, we show that abolishing one potential interaction, the A-rich loop: tRNALys3 anticodon interaction in the HIV-1 MAL strain, led to a decrease in viral infectivity and reduced the synthesis of reverse transcription products in newly infected cells. In vitro biophysical and functional experiments revealed that disruption of the extended interaction resulted in an increased affinity for reverse transcriptase (RT) and enhanced primer extension efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), vRNA was degraded by the RNaseH activity of RT, and the degradation rate was slower in the complex with the extended interaction. Consistently, the loss of vRNA integrity was detected in virions containing A-rich loop mutations. Similar results were observed in the HIV-1 NL4.3 strain, and we show that the nucleocapsid (NC) protein is necessary to promote the extended vRNA: tRNALys3 interactions in vitro. In summary, our data revealed that the additional intermolecular interaction between tRNALys3 and vRNA is likely a conserved mechanism among various HIV-1 strains and protects the vRNA from RNaseH degradation in mature virions.

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